The vagus nerve is a major conduit between lung and brain required for normal respiration. Within the airways, vagal sensory neurons detect mechanical stretch of the lung during tidal breathing, cues associated with inflammation and illness, and irritants that in some species evoke cough. However, molecular mechanisms by which vagal sensory neurons detect and encode respiratory stimuli remain poorly understood. In preliminary data, we used a molecular and genetic approach to classify sensory afferents in the airways, and adapted genetic tools to map, image, ablate, and functionally control vagal sensory neurons. We genetically tagged two sparse populations of vagal afferents (P2RY1, NPY2R) that exert powerful and opposing effects on breathing (Cell, 2015). P2RY1 neurons are largely fast-conducting a fibers that innervate clusters of pulmonary endocrine cells termed neuroepithelial bodies. Optogenetic stimulation of vagal P2RY1 neurons stops breathing, trapping animals in exhalation, without acutely impacting heart rate or gastric pressure, which are also under vagal control. NPY2R neurons are largely capsaicin-responsive C fibers, and optogenetic activation of vagal NPY2R neurons causes rapid and shallow breathing. Based on these results, we hypothesize that vagal P2RY1 neurons mediate the Hering-Breuer inspiratory reflex, while vagal NPY2R neurons are involved in pulmonary defense. These findings raise basic questions regarding the sensory stimuli in the airways that activate P2RY1 and NPY2R neurons, whether these neurons are required for normal respiration, and how these neurons sense and transduce airway cues. We will use P2ry1-ires-Cre and Npy2r-ires-Cre mice, and genetic approaches for in vivo imaging, neuron ablation, and cell-specific gene knockout to probe the sensory biology of vagal P2RY1 and NPY2R neurons. In Aim 1, we developed a new in vivo imaging paradigm in vagal ganglia that involves a genetically encoded calcium indicator, and will use this technique to query the specific response properties of vagal P2RY1 and NPY2R neurons. In Aim 2, we will selectively eliminate P2RY1 and NPY2R neurons by controlled diphtheria toxin-mediated cell ablation and determine the impact on respiratory physiology. In Aim 3, we will explore the roles of particular cell surface receptors in vagal afferents using knockout mice that lack Piezo2 or P2RY1 in some or all vagal sensory neurons. Piezo2 is abundantly expressed in a cohort of airway- innervating sensory neurons, and is a prime candidate to mediate an aspect of airway mechanosensation. Together, these studies should provide insights into cellular mechanisms underlying activation and modulation of breathing control pathways by peripheral cues. Understanding the sensory biology of respiratory control neurons in the vagus nerve may provide therapeutic targets for airway disease intervention.